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Regeneration of orofacial tissues is hampered by the lack of adequate vascular supply. Implantation of in vitro engineered, prevascularized constructs has emerged as a strategy to allow the rapid vascularization of the entire graft. Given the angiogenic properties of dental pulp stem cells, we hereby established a preclinical model of prevascularized constructs loaded with stem cells from human exfoliating deciduous teeth (SHED) in a 3-dimensional-printed material and provided a functional analysis of their in vivo angiogenesis, vascular perfusion, and permeability. Three different cell-loaded collagen hydrogels (SHED–human umbilical vein endothelial cell [HUVEC], HUVEC with SHED-conditioned medium, and SHED alone) were cast in polylactic acid (PLA) grids and ectopically implanted in athymic mice. At day 10, in vivo positron emission tomography (PETscan) revealed a significantly increased uptake of radiotracer targeting activated endothelial cells in the SHED-HUVEC group compared to the other groups. At day 30, ex vivo micro–computed tomography imaging confirmed that SHED-HUVEC constructs had a significantly increased vascular volume compared to the other ones. Injection of species-specific lectins analyzed by 2-photon microscopy demonstrated blood perfusion of the engineered human vessels in both prevascularized groups. However, in vivo quantification showed increased vessel density in the SHED-HUVEC group. In addition, coinjection of fluorescent lectin and dextran revealed that prevascularization with SHED prevented vascular leakage, demonstrating the active role of SHED in the maturation of human-engineered microvascular networks. This preclinical study introduces a novel PLA prevascularized and implantable construct, along with an array of imaging techniques, to validate the ability of SHED to promote functional human-engineered vessels, further highlighting the interest of SHED for orofacial tissue engineering. Furthermore, this study validates the use of PETscan for the early detection of in vivo angiogenesis, which may be applied in the clinic to monitor the performance of prevascularized grafts. [ABSTRACT FROM AUTHOR]

Cissus quadrangularis (C. quadrangularis) is a plant of the Vitaceae family known for its anticonvulsant effects in traditional medicine. The objective of this study was to elucidate the anxiolytic and antiepileptic effects of aqueous extract of C. quadrangularis. The mice were divided into different groups and treated for seven consecutive days as follows: a negative control group that received distilled water, po, four test groups that received four doses of the plant (37.22, 93.05, 186.11, and 372.21 mg/kg, po), and a positive control group that received sodium valproate (300 mg/kg, ip). One hour after the first treatment (first day), epilepsy was induced by intraperitoneal administration of a single dose of pilocarpine (360 mg/kg). On the seventh day, the anxiolytic effects of the extract were evaluated in the epileptic mice using the elevated plus maze (EPM) and open field (OP) paradigms. Antioxidant activities and the involvement of gabaergic neurotransmission were determined by measuring the levels of malondialdehyde, reduced glutathione (GSH), GABA, and GABA-transaminase (GABA-T) in the hippocampus of sacrificed epileptic mice. The results show that the extract of C. quadrangularis significantly and dose-dependently increased the latency to clonic and generalized tonic–clonic seizures and decreased the number and duration of seizures. In the EPM, the extract of C. quadrangularis significantly increased the number of entries and the time spent into the open arms and reduced the number of entries and the time spent into the closed arms as well as the number of rearing. The extract of C. quadrangularis also increased the number of crossing, and the time spent in the center of the OP. The level of MDA and the activity of GABA-T were significantly decreased by the extract of C. quadrangularis while reduced GSH and GABA levels were increased. The results suggest that the anticonvulsant activities of C. quadrangularis are accompanied by its anxiolytics effects. These effects may be supported by its antioxidant properties and mediated at least in part by the GABA neurotransmission.

Alzheimer’s disease the most common form of dementia in the elderly is a neurodegenerative disease that affects 44 millions of people worldwide. The first treatments against Alzheimer’s disease are acetylcholinesterase inhibitors; however, these medications are associated with many side effects. Dichrocephala integrifolia is a traditional herb widely used by indigenous population of Cameroon to treat and prevent Alzheimer’s disease and for memory improvement. In this study, we evaluated the effect of the decoction prepared from leaves of D. integrifolia, on scopolamine-induced memory impairment in mice. Seven groups of six animals were used. The first two groups received distilled water for the distilled water and scopolamine groups. The four test groups received one of the four doses of the decoction of the plant (35, 87.5, 175 or 350 mg/kg p.o.) and the positive control group received tacrine (10 mg/kg), a cholinesterase inhibitor used in the treatment of Alzheimer’s disease, during 10 consecutive days. Scopolamine (1 mg/kg), a cholinergic receptor blocker, administered 30 min after treatments, was used to induce memory impairment to all groups except the distilled water group on day 10 of drug treatment. The behavioral paradigms used to evaluate the effects of the treatment were the elevated plus maze for learning and memory, Y maze for spatial short-term memory, the novel object recognition for recognition memory and Morris water maze for the evaluation of spatial long-term memory. After behavioral tests, animals were sacrificed and brains of a subset were used for the assessment of some biomarkers of oxidative stress (malondialdehyde and reduced glutathione levels) and for the evaluation of the acetylcholinesterase activity. From the remaining subset brains, histopathological analysis was performed. The results of this study showed that, D. integrifolia at the doses of 87.5 and 350 mg/kg significantly (p < 0.01) improved spatial short-term and long-term memory, by increasing the percentage of spontaneous alternation in the Y maze and reducing the escape latency in the Morris water maze. Furthermore, the results of histopathological evaluation showed that D. integrifolia attenuated the neuronal death in the hippocampus induced by scopolamine. The main finding of this work is that D. integrifolia improves learning capacities and counteracts the memory impairment induced by scopolamine. Thus, D. integrifolia can be a promising plant resource for the management of Alzheimer’s disease and memory loss.

In this study, we investigated antiepileptogenic and neuroprotective effects of the aqueous extract of Pergularia daemia roots (PDR) using in vivo and in vitro experimental models. In in vivo studies, status epilepticus caused by pilocarpine injection triggers epileptogenesis which evolves during about 1–2 weeks. After 2 h of status epilepticus, mice were treated during the epileptogenesis period for 7 days with sodium valproate and vitamin C (standards which demonstrated to alter epileptogenesis), or Pergularia daemia. The animals were then, 1 week after status epilepticus, challenged with acute pentylenetetrazole (PTZ) administration to test behaviorally the susceptibility to a convulsant agent of animals treated or not with the plan extract. Memory was assessed after PTZ administration in the elevated plus maze and T-maze paradigms at 24 and 48 h. Antioxidant and acetylcholinesterase activities were determined in the hippocampus after sacrifice, in vitro studies were conducted using embryonic rat primary cortical cultures exposed to L-glutamate. Cell survival rate was measured and apoptotic and necrotic cell death determined. The results showed that chronic oral administration of PDR significantly and dose-dependently increased the latency to myoclonic jerks, clonic seizures and generalized tonic–clonic seizures, and the seizure score. In addition, PDR at all doses (from 4.9 to 49 mg/kg) significantly decreased the initial and retention transfer latencies in the elevated plus maze. Interestingly PDR at the same doses significantly increased the time spent and the number of entries in T-maze novel arm. PDR significantly increased the activities of acetylcholinesterase and antioxidant enzymes superoxide dismutase, catalase, and total glutathione and proteins, and decreased malondialdehyde level. Furthermore, PDR increased viability rate of primary cortical neurons after L-glutamate-induced excitotoxicity, in a dose dependent manner. Altogether these results suggest that PDR has antiepileptogenic and neuroprotective effects, which could be mediated by antioxidant and antiapoptotic activities.

Abstract Background Infant mortality rates (IMR) remain high in many sub-Saharan African countries, especially in rural settings where access to health services may be limited. Studies in such communities can provide relevant data on the burden of and risk factors for infant death. We measured IMR and explored risk factors for infant death in a cohort of children born in Banfora Health District, a rural area in South-West Burkina Faso. Methods A prospective community-based cohort study was nested within the PROMISE-EBF trial (NCT00397150) in 24 villages of the study area. Maternal and infant baseline characteristics were collected at recruitment and after birth, respectively. Home visits were conducted at weeks 3, 6, 12, 24 and 52 after birth. Descriptive statistics were calculated using robust standard errors to account for cluster sampling. Cox multivariable regression was used to investigate potential risk factors for infant death. Results Among the 866 live born children included in the study there were 98 infant deaths, yielding an IMR of 113 per 1000 live births (95% CI: 89–143). Over 75% of infant deaths had occurred by 6 months of age and the post neonatal infant mortality rate was 67 per 1000 live births (95% CI: 51–88). Infections (35%) and preterm births complications (23%) were the most common probable causes of death by 6 months. Multivariable analyses identified maternal history of child death, polygyny, twin births and poor anthropometric z-scores at week-3 as factors associated with increased risk of infant death. Conclusions We observed a very high IMR in a rural area of Burkina Faso, a country where 75% of the population lives in rural settings. Community-based health interventions targeting mothers and children at high risk are urgently needed to reduce the high burden of infant deaths in these areas.

Cissus quadrangularis (C. quadrangularis) is a plant of the Vitaceae family known for its anticonvulsant effects in traditional medicine. The objective of this study was to elucidate the anxiolytic and antiepileptic effects of aqueous extract of C. quadrangularis. The mice were divided into different groups and treated for seven consecutive days as follows: a negative control group that received distilled water, po, four test groups that received four doses of the plant (37.22, 93.05, 186.11, and 372.21 mg/kg, po), and a positive control group that received sodium valproate (300 mg/kg, ip). One hour after the first treatment (first day), epilepsy was induced by intraperitoneal administration of a single dose of pilocarpine (360 mg/kg). On the seventh day, the anxiolytic effects of the extract were evaluated in the epileptic mice using the elevated plus maze (EPM) and open field (OP) paradigms. Antioxidant activities and the involvement of gabaergic neurotransmission were determined by measuring the levels of malondialdehyde, reduced glutathione (GSH), GABA, and GABA-transaminase (GABA-T) in the hippocampus of sacrificed epileptic mice. The results show that the extract of C. quadrangularis significantly and dose-dependently increased the latency to clonic and generalized tonic–clonic seizures and decreased the number and duration of seizures. In the EPM, the extract of C. quadrangularis significantly increased the number of entries and the time spent into the open arms and reduced the number of entries and the time spent into the closed arms as well as the number of rearing. The extract of C. quadrangularis also increased the number of crossing, and the time spent in the center of the OP. The level of MDA and the activity of GABA-T were significantly decreased by the extract of C. quadrangularis while reduced GSH and GABA levels were increased. The results suggest that the anticonvulsant activities of C. quadrangularis are accompanied by its anxiolytics effects. These effects may be supported by its antioxidant properties and mediated at least in part by the GABA neurotransmission. [ABSTRACT FROM AUTHOR]

Alzheimer's disease the most common form of dementia in the elderly is a neurodegenerative disease that affects 44 millions of people worldwide. The first treatments against Alzheimer's disease are acetylcholinesterase inhibitors; however, these medications are associated with many side effects. Dichrocephala integrifolia is a traditional herb widely used by indigenous population of Cameroon to treat and prevent Alzheimer's disease and for memory improvement. In this study, we evaluated the effect of the decoction prepared from leaves of D. integrifolia, on scopolamineinduced memory impairment in mice. Seven groups of six animals were used. The first two groups received distilled water for the distilled water and scopolamine groups. The four test groups received one of the four doses of the decoction of the plant (35, 87.5, 175 or 350 mg/kg p.o.) and the positive control group received tacrine (10 mg/kg), a cholinesterase inhibitor used in the treatment of Alzheimer's disease, during 10 consecutive days. Scopolamine (1 mg/kg), a cholinergic receptor blocker, administered 30 min after treatments, was used to induce memory impairment to all groups except the distilled water group on day 10 of drug treatment. The behavioral paradigms used to evaluate the effects of the treatment were the elevated plus maze for learning and memory, Y maze for spatial short-term memory, the novel object recognition for recognition memory and Morris water maze for the evaluation of spatial long-term memory. After behavioral tests, animals were sacrificed and brains of a subset were used for the assessment of some biomarkers of oxidative stress (malondialdehyde and reduced glutathione levels) and for the evaluation of the acetylcholinesterase activity. From the remaining subset brains, histopathological analysis was performed. The results of this study showed that, D. integrifolia at the doses of 87.5 and 350 mg/kg significantly (p < 0.01) improved spatial short-term and long-term memory, by increasing the percentage of spontaneous alternation in the Y maze and reducing the escape latency in the Morris water maze. Furthermore, the results of histopathological evaluation showed that D. integrifolia attenuated the neuronal death in the hippocampus induced by scopolamine. The main finding of this work is that D. integrifolia improves learning capacities and counteracts the memory impairment induced by scopolamine. Thus, D. integrifolia can be a promising plant resource for the management of Alzheimer's disease and memory loss. [ABSTRACT FROM AUTHOR]

In this study, we investigated antiepileptogenic and neuroprotective effects of the aqueous extract of Pergularia daemia roots (PDR) using in vivo and in vitro experimental models. In in vivo studies, status epilepticus caused by pilocarpine injection triggers epileptogenesis which evolves during about 1-2 weeks. After 2 h of status epilepticus, mice were treated during the epileptogenesis period for 7 days with sodium valproate and vitamin C (standards which demonstrated to alter epileptogenesis) or Pergularia daemia. The animals were then, 1 week after status epilepticus, challenged with acute pentylenetetrazole (PTZ) administration to test behaviorally the susceptibility to a convulsant agent of animals treated or not with the plan extract. Memory was assessed after PTZ administration in the elevated plus maze and T-maze paradigms at 24 and 48 h. Antioxidant and acetylcholinesterase activities were determined in the hippocampus after sacrifice, in vitro studies were conducted using embryonic rat primary cortical cultures exposed to L-glutamate. Cell survival rate was measured and apoptotic and necrotic cell death determined. The results showed that chronic oral administration of PDR significantly and dose-dependently increased the latency to myoclonic jerks, clonic seizures and generalized tonic-clonic seizures and the seizure score. In addition, PDR at all doses (from 4.9 to 49 mg/kg) significantly decreased the initial and retention transfer latencies in the elevated plus maze. Interestingly PDR at the same doses significantly increased the time spent and the number of entries in T-maze novel arm. PDR significantly increased the activities of acetylcholinesterase and antioxidant enzymes superoxide dismutase, catalase, and total glutathione and proteins, and decreased malondialdehyde level. Furthermore, PDR increased viability rate of primary cortical neurons after L-glutamate-induced excitotoxicity, in a dose dependent manner. Altogether these results suggest that PDR has antiepileptogenic and neuroprotective effects, which could be mediated by antioxidant and antiapoptotic activities. [ABSTRACT FROM AUTHOR]

Introduction: Infra-popliteal angioplasty continues to be widely performed with minimal evidence to guide practice. Endovascular device selection is contentious and there is even uncertainty over which artery to treat for optimum reperfusion. Direct reperfusion (DR) targets the artery supplying the ischaemic tissue. Indirect reperfusion (IR) targets an artery supplying collaterals to the ischaemic area. Our unit practice for the last eight years has been to attempt to open all tibial arteries at the time of angioplasty. When successful, this results in both direct and indirect; or combined reperfusion (CR). The aim was to review the outcomes of CR and compare them with DR or IR alone. Methods: An eight year retrospective review from a single unit of all infra-popliteal angioplasties was undertaken. Wound healing, limb salvage, amputation-free and overall survival data as well as re-intervention rates were captured for all patients. Subgroup analysis for diabetics was undertaken. Kaplan Meier curves are presented for survival outcomes. All odds and hazard ratios (HR) and p values were corrected for bias from confounders using multivariate analysis. Results: 250 procedures were performed: 22 (9%) were CR; 115 (46%) DR and 113 (45%) IR. Amputation-free survival (HR 0.504, p = 0.039) and re-intervention and amputation-free survival (HR 0.414, p = 0.005) were significantly improved in patients undergoing CR compared to IR. Wound healing was similarly affected by reperfusion strategy (OR = 0.35, p = 0.047). Effects of CR over IR were similar when only diabetic patients were considered. Conclusions: Combined revascularisation can only be achieved in approximately 10% of patients. However, when successful, it results in significant improvements in wound healing and amputation-free survival over simple indirect reperfusion techniques. [ABSTRACT FROM AUTHOR]

Indoor air quality was characterized in 10 recently built energy-efficient French schools during two periods of 4.5 days. Carbon dioxide time-resolved measurements during occupancy clearly highlight the key role of the ventilation rate (scheduled or occupancy indexed), especially in this type of building, which was tightly sealed and equipped with a dual-flow ventilation system to provide air refreshment. Volatile organic compounds ( VOCs) and inorganic gases (ozone and NO2) were measured indoors and outdoors by passive techniques during the occupied and the unoccupied periods. Over 150 VOC species were identified. Among them, 27 species were selected for quantification, based on their occurrence. High concentrations were found for acetone, 2-butanone, formaldehyde, toluene, and hexaldehyde. However, these concentrations are lower than those previously observed in conventional school buildings. The indoor/outdoor and unoccupied/occupied ratios are informative regarding emission sources. Except for benzene, ozone, and NO2, all the pollutants in these buildings have an indoor source. Occupancy is associated with increased levels of acetone, 2-butanone, pentanal, butyl acetate, and alkanes. [ABSTRACT FROM AUTHOR]

Supraglacial streams are a significant part of the glacial hydrological system and important for understanding the connection between glacial hydrology and glacier dynamics. Here we determine the factors that influence the development of step-pool formation and pulsating flow in a supraglacial stream on Bylot Island, Nunavut. Results show that during the second week of a 2-week study, multiple successive rainfall events occurred, stream temperature increased and ablation decreased; which also caused stream discharge to decrease. In addition, the stream, which flowed over a 13 m high waterfall off the front of Fountain Glacier, rapidly formed 21 step-pools and began to pulsate. The pulsating phenomenon involved the complete stoppage of flow over the waterfall and the subsequent restart between 8 and 20 s later. Pulsating flow resulted from rapid changes in the streambed morphology. In particular, the formation of the step-pool sequence was caused by helical flow around meander bends and hydrologically induced slippage along transverse shear planes, evidenced by observations of high-pressure artesian flow from transverse fractures. Contrary to previous literature, this study shows that high discharge is not necessarily the cause of step-pool formation and pulsating flow within supraglacial streams. [ABSTRACT FROM PUBLISHER]

Aim The aim of this study is to determine if radiographic features are predictive of surgical difficulty of open exposure of palatal canines using a tissue sacrifice method. Materials and methods This is a prospective case series comparing features observed on preoperative panoramic radiographs and other variables to both surgical duration and the surgeons reported difficulty. Radiographic features included height, sector (overlap of adjacent incisor) and angulation of the impacted canine. Clinical variables recorded were patient age and gender, side of impaction, anaesthetic choice, if the canine was palpable, and patient cooperation. A stepwise linear regression analysis was used to relate clinical and radiographic variables to treatment duration and assessed difficulty. Inter- and intra-examiner reliability were assessed with Kappa statistics. Results Forty-nine patients with a total of 55 palatal canine exposures were included. The mean duration of exposure was 4 min and 5 s with a range of 45 s to 8 min and 50 s. Surgical duration was correlated to a subjective grade of surgical difficulty recorded on a visual analogue scale by the surgeon ( Pearson's correlation coefficient 0.76). Three predictors of increased surgical duration were identified as anaesthetic choice, height and angulation of impaction. The linear regression had an accuracy of 30.5%. Conclusions Radiographic features had a small predictive value in determining duration of open palatal canine exposure. Both increased height and angulation to the midline were associated with longer procedures. The use of local anaesthetic was associated with a longer surgical duration compared with general anaesthetic. [ABSTRACT FROM AUTHOR]

Women with antiphospholipid syndrome (APS) may have diverse pregnancy outcomes. The objective of this study was to evaluate pregnancy outcome in women with APS according to their clinical phenotype, i.e. thrombotic and obstetric APS. Eighty-three pregnancies in 67 women with APS were included in the study, including 21 with recurrent miscarriage (Group 1), 21 with late fetal loss or early delivery due to placental dysfunction (Group 2) and 41 with thrombotic APS (Group 3). Group 3 had higher rates of preterm delivery (26.8% versus 4.7%, p=0.05) than Group 1 and more small for gestational age (SGA) babies than Group 2 (39.5% versus 4.8%, p=0.003). Group 2 had significantly longer gestations compared with their pretreatment pregnancies (38.4 [28.4-41.4] versus 24.0 [18-35] weeks, p<0.0001) and 100% live birth rate after treatment with aspirin and low-molecular-weight heparin (LMWH). In conclusion, women with thrombotic APS (Group 3) have higher rates of pregnancy complications than those with obstetric APS (Groups 1 and 2). Treatment with aspirin and LMWH is associated with improved outcomes for women with previous late fetal loss or early delivery due to placental dysfunction (Group 2). [ABSTRACT FROM AUTHOR]

Management of pregnant women with renal disease involves awareness of, and allowance for, physiological changes including decreased serum creatinine and increased proteinuria. For women with systemic lupus erythematosus (SLE), pregnancy increases likelihood of flare. These can occur at any stage, and are more difficult to diagnose, as symptoms overlap those of normal pregnancy. Renal involvement is no more common in pregnancy. Worsening proteinuria may be lupus flare but differential includes pre-eclampsia. In women with chronic renal disease, pregnancy may accelerate decline in renal function and worsen hypertension and proteinuria, with increased risk of maternal (eg, pre-eclampsia) and fetal (eg, IUGR, IUD) complications, strongly correlating with degree of renal impairment peri-conception. Pregnancy success rate varies from 20% to 95% depending on base-line creatinine. Best outcome is obtained if disease was quiescent for >6 months pre-conception. Women on dialysis or with renal transplants can achieve successful pregnancy but have higher maternal and fetal complication rates. Acute on chronic renal failure can develop secondary to complications such as HELLP and AFLP. Management needs to be by a multidisciplinary team involving physicians and obstetricians, ideally beginning with pre-pregnancy counselling. Treatment of flares includes corticosteroids, hydroxychloroquine, azothioprine, NSAIDs and MMF. Blood pressure is controlled with methyldopa, nifedipine or hydralazine. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the effect of vascular occlusion on radio-frequency (RF) lesion size and on potential associated biliary and portal lesions. Radio-frequency lesions using a 1-cm exposed-tip cooled electrode were created in pig liver. Liver perfusion was modified by arterial embolization (n=2), left portal clamping (n=2), and both (n=2). Two pigs were used as controls. Two weeks after, control portography was performed, animals were killed, and ex-vivo cholangiography was carried out. Pathological studies evaluated the lesion surface and associated portal and biliary damages. A mathematical regression model showed that portal occlusion increased by 43 mm2 (+40%) the surface of RF lesions, arterial occlusion by 135 mm2 (+126%), and associated occlusion by 466 mm2 (+435%). Biliary stenoses were found in 4 cases (two arterial occlusions, one portal occlusion, and one associated occlusion). One case of partial portal vein thrombosis was found in one case of portal occlusion and resolved at 2 weeks. Ischemic damages adjacent to RF lesions were found in cases of combined occlusions. The reduction of liver perfusion increases significantly the size of RF lesions but is associated with a risk of biliary, portal, or parenchymal complications. [ABSTRACT FROM AUTHOR]

Although capillary leakage is a major feature of systemic inflammatory response syndrome (SIRS) associated with shocks, molecular mechanisms remain uncharacterized in humans. Patients with severe cardiogenic shock were recruited from La Pitié-Salpêtrière intensive care unit (ICU) and RNAseq analysis of circulating monocytes was performed. Thirty-eight genes associated with capillary leakage were thus identified. Beyond them, amphiregulin is a ligand of EGF receptor involved in angiogenesis, and its expression is triggered by lipopolysaccharides in monocytes. We first confirmed association between AMPHIREGULIN plasma level and vascular leakage in a validation cohort of 77 cardiogenic shock patients. We here hypothesize that AMPHIREGULIN may play a role in SIRS-induced capillary leakage. The aim of the project is to characterize its mechanisms of action. Vascular permeability was compared in amphiregulin-KO or WT-mice using a model of LPS-induced capillary leakage. Total/dry weight ratio (W/D ratio) of lungs, heart, liver and kidney as well as quantification of Evans blue leakage were used to quantify vascular permeability. In two preliminary experiments (n = 15 KO and 9 WT), lung W/D ratio of amphiregulin-KO mice was significantly reduced compared to WT-mice after LPS injection (median 4.27 [IQR 4.16-4.42] vs. 4.53 [4.41-4.61], P < 0.01). A similar trend was quantified in the liver (3.38 [3.3-3.49] vs. 3.52 [3.38-3.56], P = 0.096). In contrast, W/D ratio in other organs were not affected. These preliminary results indicate that AMPHIREGULIN may be an important regulator of vascular permeability in SIRS induced by cardiogenic shock. Further experiments are presently ongoing to confirm these results. [ABSTRACT FROM AUTHOR]

L’ARN interférence, mécanisme de régulation de l’expression des gènes, est médiée par les siARNs et les microARNs, ARN non-codants de 20 à 22 nucléotides affectant la régulation post-transcriptionnelle d’ARNm cibles avec lesquels ils s’apparient. La RNase DICER est une enzyme centrale de la biosynthèse des siARNs et microARNs. Les souris dont le gène dicer est invalidé ont un phénotype complexe, et meurent très tôt pendant le développement, notamment à cause d’un défaut d’angiogenèse. Afin d’étudier l’ARN interférence au cours de l’angiogenèse embryonnaire, des souris dont le gène dicer est floxé (mutant conditionnel) sont croisées avec des souris exprimant la recombinase Cre, de manière constitutive, sous le contrôle du promoteur du gène tie2, dirigeant ainsi son expression dans les cellules endothéliales (CE) et les cellules hématopiétiques. Nos résultats montrent que l’invalidation de dicer sous le contrôle du promoteur du gène tie2 entraine une mortalité embryonnaire suite à un œdème et des hémorragies au treizième jour du développement (E13,5). L’analyse histologique montre des vaisseaux lymphatiques remplis de sang, suggérant une mauvaise séparation du réseau sanguin et lymphatique. Cette hypothèse est étudiée par marquage des vaisseaux lymphatiques (LYVE-1) et des vaisseaux sanguins (PECAM) sur embryon entier et peaux isolées à différents stades précédant la mort. Ces embryons présentent également un problème de développement du foie, probablement dû à l’activité du promoteur tie2 dans les lignées hématopoiétiques. La mise en culture de ces foies fœtaux à E13,5 révèle une atteinte des précurseurs hématopoétiques. L’étude de ces précurseurs à des stades plus précoces (E8,5) est en cours au laboratoire. Nos résultats démontrent donc un rôle important de l’ARN interférence dans le contrôle épigénétique de l’angiogenèse et de la lymphangiogenèse embryonnaire mais également dans le développement de l’hématopoièse, suggérant son implication dans la différenciation veino-lymphangiogenèse, dont les mécanismes moléculaires seront discutés. [Copyright &y& Elsevier]

De nombreux facteurs épigénétiques, dont l’ARN interférence module l’expression génique au cours des pathologies cardiovasculaires. Bien que les micro-ARNs (miARN) soient exprimés dans le système cardiovasculaire, leur rôle est jusqu’alors peu connu. L’objectif de ce travail est d’étudier le rôle de l’ARN interférence dans la régulation de l’angiogenèse développementale et pathologique par invalidation de DICER in vivo spécifiquement dans les cellules musculaires lisses (CML). Nous disposons de souris dont le gène dicer est floxé (allèle dicer conditionnel) 1 [1] Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ: The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb, Proc Natl Acad Sci U S A 2005, 102:10898-10903. , permettant d’invalider spécifiquement Dicer dans les cellules exprimant une recombinase Cre sous le contrôle de promoteurs spécifiques des cellules musculaires lisses (α-SMA ; SM-MHC ; sm22), Ces souris ont aussi été croisées avec des souris R26R permettant d’analyser les cellules recombinées 2 [2] Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain, Nat Genet 1999, 21:70-71. . Nos premiers résultats montrent que les embryons dont le gène dicer sous le contrôle du promoteur sm22 présentent des anomalies de développement embryonnaire à E16,5 (figure 1). Les embryons mutants souffrent d’hémorragies importantes entraînant la mort. La mort embryonnaire ne correspond à priori pas à des défauts de vasculogenèse importants, mais est plutôt caractéristique de défauts de remodelage angiogénique tels qu’une perméabilité endothéliale exacerbée ou une mauvaise différenciation des péricytes, induisant des vaisseaux immatures et perméables. Nous analysons donc les défauts vasculaires responsables de la mort embryonnaire en étudiant la morphologie des vaisseaux sanguins des souris mutantes, au cours du développement, par marquage spécifique des veines, des artères, des CML ou des péricytes et des vaisseaux lymphatiques par marquages immunohistochimiques CD31, α-SMA, NG2, et VEGF-R3. Display Omitted [Copyright &y& Elsevier]

Abdominal aortic aneurysms (AAA) are the third cause of cardiovascular mortality in industrialized countries. AAA consist in progressive dilatation of the aorta which can lead to spontaneous rupture. Aneurysm evolution depends on the balance between degradation and repair of elastin and collagens in the aortic extracellular matrix (ECM), due to inflammatory cells infiltration in parallel with loss of vascular smooth muscle cells (VSMC). The cross-linking enzymes, lysyl oxidase (LOX), lysyl oxidase-like protein 2 (LOXL2) and transglutaminase 2 (TG2) are involved in the maturation of ECM components and arterial remodeling, by covalently bonding elastin and collagens. These crosslinking enzymes are therefore good candidates for AAA stabilisation and prevention of rupture, through ECM remodeling. We show that TG2 and LOXL2 are expressed in endothelial cells and VSMC, in addition to LOX, as they were detected in the intima and the media of healthy mouse aortic wall by immunofluorescence. In addition, these enzymes are all present in the ECM Triton-resistant fraction. We used a murine CaCl2-induced aneurysm model, in which AAA progression is associated with a decrease in VSMC density, as demonstrated by smooth muscle alpha-actin immunostaining. Whereas LOX and TG2 expression was strongly decreased in parallel with the loss of VSMC, LOXL2 expression was less severely affected. The involvement of lysyl oxidases in AAA was investigated using the irreversible inhibitor, beta-aminopropionitrile (BAPN). After induction of AAA by CaCl2, BAPN-treated mice showed a greater increase of the internal aortic diameter in comparison with untreated controls, suggesting that activity from remaining lysyl oxidase partially prevents AAA progression. In parallel, fibrillar collagens content is determined by Sirius red staining and second harmonic generation imaging; and elastin fibers organisation by Orcein-staining. These ongoing experiments will characterize the involvement of crosslinking enzymes in ECM repair during the evolution of AAA. Altogether, this study will allow to determine whether LOXL2 and/or TG2 could be therapeutic targets for stabilizing AAA progression through ECM repair. [Copyright &y& Elsevier]

Backround: Angiopoietin-like 4 (ANGPTL4) is a secreted protein of the Angiopoietins family involved in angiogenesis and vessel maintenance. We identified angptl4 as a hypoxia induced gene in endothelial cells in vitro. In humans, ANGPTL4 is expressed in hypoxic areas such as vascular cells of human critical leg ischemia samples. In the heart, focal expression in myocytes, vessels and infiltrating monocytes was also evidenced in infarcted areas. Material & Methods: We used C57/Bl6 angptl4 knock-out mice (Regeneron pharmaceuticals, Tarrytown). The pattern of expression of angptl4 was determined during development using Lac Z staining allowed by the insertion of a beta-galactosidase cassette. At the adult stage, myocardial infarction was induced by ligation of the left coronary artery for 45mn. The hearts were analyzed 24 h, 48 h and 2 weeks after reperfusion. Results: During embryonic development, angptl4 is expressed in endothelial cells from intersomitic, limb bud (E10,5-E11,5) and heart vessels (E12,5-E14,5). At later stages (from E15,5), angptl4 is also expressed in skin vessels. Angptl4 knock-out mice showed an increased infarcted area compared to wild type littermates mice (+31 %, p=0.01) 48h after reperfusion, suggesting a cardioprotective role of ANGPTL4. Immunohistological analyzes revealed dramatic tissue damages in the angptl4 knock-out mice in term of oedema, hemorragies and necrosis correlated with an increased circulating monocytes infiltration (+36 %, p=0.004). PECAM-1 stainings revealed morphologically modified angiogenic capillaries in angptl4 KO mice compared to controls suggesting a modulated vascular permeability. No difference was observed between isolated cardiomyocytes from both groups submitted to a survival assay under hypoxia. Conclusion: Angptl4 is expressed by endothelial cells during embryonic and during adult stage following hypoxic stress. Experiments performed using the ischemia-reperfusion model show that ANGPTL4 has a cardioprotective role modulating both endothelium integrity and post-ischaemic inflammation. [Copyright &y& Elsevier]

Hypoxia stimulates angiogenesis during development, as well as in the course of ischemic cardiovascular diseases and tumor growth. In a hypoxic environment, endothelial cells (EC) are key players of the angiogenic response. Their activation leads to remodeling of the extracellular matrix (ECM): a degradation step generates a provisional ECM supporting EC proliferation and migration; assembly of a new basal lamina leads to pericyte recruitment and neovessel maturation. In order to identify endothelial ECM proteins regulated by hypoxia, 2D gel electrophoresis was performed on ECM samples prepared from cultured EC of micro or large vessels (HDMEC or HUVEC respectively). Mass spectrometry identified lysyl oxidase-like protein 2 (LOXL2) as a highly hypoxia-induced protein. Lysyl oxidases are secreted enzymes involved in ECM maturation through covalent cross-linking of its major components, collagens and elastin. The induction of LOXL2 expression was detected both at the mRNA and protein levels. Using siRNA, we demonstrated that LOXL2 is responsible for 65 % of lysyl oxidase total activity in hypoxic EC. In addition, LOXL2 protein was colocalised with type IV collagen in endothelial ECM. We further investigated the expression of LOXL2 in vivo. The enzyme was expressed in rat EC from retina during postnatal vascular development, and from adult skeletal muscle. In a murine model of hindlimb ischemia, LOXL2 was upregulated at the protein and mRNA levels. In situ hybridization revealed its expression in both EC and macrophages. Involvement of LOXL2 at different steps of the angiogenic process was further studied in vitro. We demonstrated that LOXL2 increases EC migration on fibronectin, as well as migration and tube formation in fibrin 3D gels. In addition, LOXL2 knock-down inhibited type IV collagen deposition in the ECM, suggesting a major role for LOXL2 in the organisation of endothelial basal lamina. Finally, whereas the efficiency of endothelial ECM for recruiting VSMC was increased by hypoxia, this effect was reduced upon knocking down endothelial LOXL2 expression. Altogether, these data suggest that LOXL2 plays a major role in EC migration, vascular basal lamina deposition and neovessel stabilisation during hypoxia-induced angiogenesis. [Copyright &y& Elsevier]

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells. It is highly expressed in tumor cells from conventional renal carcinoma and in hypoxic perinecrotic areas of numerous types of human tumors (1). We previously showed that ANGPTL4, through its action on both vascular and tumor cells, prevents metastastes through inhibition of vascular permeability as well as tumor cell motility and invasiveness. In vivo, using both Lewis Lung carcinoma cells as well as melanoma B16 cells, we showed ANGPTL4 inhibits both intravasation extravasation of tumor cells. Using Miles assay, ANGPTL4 inhibited the histamine-induced vascular permeability in electrotransferred mice overexpressing ANGPTL4 compared to control mice (2). More recently, Padua et al. revealed a clinical association between TGFbeta activity in primary tumors and risk of distant recurrence, specifically for estrogen receptor negative breast tumors with lung metastasis but not bone metastasis. In vivo, lung metastasis seeding from mammary tumors depends on TGFbeta receptors, Smad function and ANGPTL4 expression (3). Both groups therefore report ANGPTL4 as a key regulator of vascular permeability. Nevertheless, better insights are needed in order to precisely characterize its role during tumor angiogenesis and subsequent metastases in various organs, namely lungs and bones. The aim of the present study is to address the in vivo role of ANGPTL4 in modulating vascular integrity, using Lewis Lung carcinoma cells xenografted in ANGPTL4 KO mice. Inhibition of tumor growth is observed in KO mice compared to WT mice (1207+/-105 mm3 versus 3053+/-569 mm3 at day 26, p=0.002). At day 33, 60 % of the angptl4 KO mice show large lung macrometastases whereas only 28 % of wild type lungs have been invaded by small micrometastases. Further experiments using different metastatic models are ongoing in order to perform imaging analysis of blood vessels and endothelium integrity the during the metastatic process in angptl4 knock-out and WT mice.1. Le Jan, S. (2003) Am J Pathol 162, 1521-8.2. Galaup, A. (2006) Proc Natl Acad Sci U S A 103, 18721-6.3. Padua, D. (2008) Cell 133, 66-77. [Copyright &y& Elsevier]

Hypoxia is a potent inducer of angiogenesis during which endothelial cells (ECs) undergo modulations of cell-cell and cell-matrix interactions. ANGPTL4 expression is induced by hypoxia in ECs (1,2) and interacts with the extracellular matrix (3) modulating ECs adhesion, migration and sprouting through cytoskeleton reorganisation (4). Objective: Analyse whether in vivo ANGPTL4 plays a role in modulating hypoxia-driven angiogenesis in the retina, both in development and in vascular retinal pathologies. Materials: C57/Bl6 angptl4 KO mice and WT littermates were used. An hyperoxia chamber was used to establish the oxygen-induced retinopathy (OIR). Mice were exposed to 75 % oxygen from P7 to P12 leading to retinal vessel loss. After returning to room air, hypoxia induces neovascularisation, evaluated at P17 when greatest. Results: Developmental angiogenesis of the retina in P5-P7 pups was analysed. Angptl4 is expressed by ECs and angptl4 -/- retinas show a decreased number of branchpoints (p<0,005) as well as an increased vascular density (p<0,001). Angptl4 -/- veins were dilated compared to angptl4+/- (p<0,001). Altogether, loss of angptl4 expression in the developing retina lead to an immature vascular plexus. Moreover, angptl4 -/- retinas showed an increased basal vascular leakage (p<0,0,05). Confocal analysis also showed that pericyte coverage might be involved. We also show that ANGPTL4 is expressed during OIR by ECs. Retinal wholemount preparations stained with isolectin B4 were performed. At P12, the retinal vaso-obliterated area was not affected in angptl4 -/- mice, suggesting that ANGPTL4 has no effect in the hyperoxia-induced vaso-obliteration. In contrast, the neovascular angiogenic response was significantly alteredin angptl4 -/- mice at P17 (p<0,001). Thus, ANGPTL4 is essential for regulating the growth of new retinal vessels in an ischemic environment. Conclusions: Altogether, this data demonstrate that ANGPTL4 plays a critical role in hypoxia-induced angiogenesis, both in developmental and pathological conditions, by affecting 1) vascular density, 2) basal vascular permeability, and 3) pericyte coverage. (1)Le Jan S, Am J Path. 2003 (2)Galaup A, Proc Natl Acad Sci U S A. 2006 (3)Cazes A, Circ Res. 2006 (4)Chomel C, FASEB J. 2008 [Copyright &y& Elsevier]